Stem Cells: Analyzing Sex

The Challenge

Biological sex is commonly studied as a variable in research with humans, but analyzing sex is rare in animal research and rarer still in cell-based research (Beery et al., 2011). This deficiency can represent a lost opportunity to understand basic and developmental biology, and to refine cell-based therapies.

Sex should be analyzed at all levels, from chromosomes and cells to whole organisms. Taking sex into account has led to novel questions about stem cells. Analyzing sex involves:

1. Designing research to use cells of both sexes in sufficient quantities to detect or rule out sex differences (not all sex differences will be significant).
2. Reporting the sex of cells used in experiments.
3. Recording, formatting, and analyzing data to allow for systematic review and meta-analysis. Reviews can identify gaps in knowledge (when, for example, experiments have involved cells of only one sex). Meta-analysis can increase statistical power and may allow sex analysis even in the absence of two-sex studies.

Gendered Innovations:

1. Identifying Sex Differences in Stem Cell Characteristics. Research using animal models has shown that the sex of stem cells may influence therapeutically relevant cell traits, such as proliferation and differentiation rates.2. Understanding Differences within and between XX and XY Stem Cells. Discoveries about interactions between genetics, hormonal environments, and epigenetics have improved the understanding of stem cell biology.3. Improving Clinical Guidelines for Stem Cell Therapies. By formulating research questions about the importance of donor and recipient sex (along with other factors that interact with sex) in hematopoietic stem cell transplantation, researchers have gathered data relevant to improving clinical guidelines for this stem cell therapy.

The Challenge

Taking sex into account can advance basic knowledge regarding stem cells—demonstrating potential sex differences in therapeutic capacity as well as sex differences in receptor-mediated pathways. Basic knowledge of stem cell biology is important to one of the most active areas of stem cell research: inducing pluripotency in cells derived from adult patients and utilizing these cells to repair or reconstruct organs.

2. Differences in Muscle-Derived Stem Cell (MDSC) Regenerative Capacity
MDSCs have the capacity for myocardial repair as well as skeletal muscle repair. They may also be useful for treating muscular dystrophy, for which existing treatments have limited effect (Jankowski et al., 2002).

MDSC cell lines display variability in regenerative ability. Using mdx mice, which spontaneously develop muscular dystrophy, Deasy et al. demonstrated that cell sex, independent of other variables such as immune response and exogenous estrogenic effects, exerts a strong effect on regenerative capacity. The mechanism behind these differences is an active area of research.

Deasy et al. found significant sex differences in regeneration capacity in vivo, with XX cells yielding a higher regeneration index (RI) than XY cells. In vivo studies took advantage of the fact that mdx mouse muscle fibers lack the protein dystrophin; researchers determined RI by quantifying muscle fibers generated from stem cells (i.e., those with dystrophin). Even though all MDSCs could differentiate into dystrophin-expressing fibers in vitro, only XX MDSCs could regenerate robustly in vivo (Deasy et al., 2007).

These sex differences may be therapeutically relevant—but because many variables besides sex influence cell behavior, and because the traits of an “ideal” cell type differ depending on the therapy in question, such differences do not indicate that cells of a given sex are broadly therapeutically superior to cells of the other sex. In clinical research using stem cells, there is a “lack [of] direct comparisons of different cell types in clearly defined, clinically relevant models of disease” (Zenovich et al., 2007).

Gendered Innovation 2: Understanding Differences within and between XX and XY Stem Cells

Knowing that sex differences exist in stem cells, researchers sought to elucidate the causes of these differences—work that required analysis of additional factors (see Method).

Researchers who analyzed sex have observed differences between XX and XY stem cells, but a deeper understanding of stem cell biology requires examination of covariates. Observed sex differences can arise in several ways:

1. Genetics: Female and male stem cells differ in karyotype and therefore differ genetically, but genetic variations also exist between cells of the same sex—not all XX cells or XY cells are alike. Studying the covariates of genotype and investigating both between-sex and within-sex differences is important in stem cell research.

2. Hormonal Environment: Stem cells are sensitive to hormonal environment—often including, but not limited to, the presence of sex hormones. Hormones can have both transient and permanent effects on stem cells, making hormonal environment a necessary covariate to sex (Asselin-Labat et al., 2010).

3. Epigenetics: The DNA sequence of a stem cell is unchanged throughout the cell’s life and is rarely altered by environment. Gene expression, however, can change frequently and dramatically; indeed, such changes account for the ability of genetically identical stem cells to differentiate into functionally distinct somatic cells. These changes are heritable, and so even if cells are cultured in vitro in identical hormonal environments, observed differences cannot be assumed to stem from genetic sequence alone. The environments in which these cells’ ancestors developed may have created epigenetic differences, and they are important covariates (Ohm et al., 2009).

Multivariate studies include sex as one variable among many. It is important to test for interactions between sex and other predictors of the outcome under study. Without such testing, one might attribute variability to sex when that difference is actually dependent on another factor. This misattribution can lead to overemphasis of sex differences. Covariate analysis has shown the following:

1. Species influences stem cell behavior, and findings in animal models are not necessarily applicable to humans. For example, when pluripotency is induced in murine XX fibroblasts, the resultant induced pluripotent stem cells (iPSCs) show a reversal of X inactivation, with two active X chromosomes. When human XX fibroblasts are treated to induce pluripotency, however, the resultant human iPSCs display one active and one inactive X chromosome (Tchieu et al., 2010).

2. In mouse models of muscular dystrophy, both the sex of the donor cell and the sex of the recipient animal matter. Multivariate analysis shows that XX MDSCs promote regeneration more than XY MDSCs (regardless of recipient sex) and that female recipient animals undergo more regeneration than male recipient animals (regardless of donor cell sex). In the mdx model of muscular dystrophy, matching donor sex to recipient sex would not be an optimal strategy for promoting muscular regeneration: XX stem cells are a better treatment option for both females and males (Deasy et al., 2007).

Further experiments using immune deficient mice suggest that the effect of host sex (but not the effect of cell sex) is immunologically modulated: Researchers “observed no significant difference as a result of host sex” in immune deficient animals, “yet the significant difference as a result of cell sex remained” (Deasy et al., 2007).

3. Even when sex is a statistically significant predictor of stem cell behavior, not all cell lines are alike within a sex. Mouse MDSCs show significant variation in regeneration potential within a single sex (Deasy et al., 2007).

Studies of the 17 human embryonic stem cell lines commonly used in research (9 of which are 46,XX and 8 of which are 46,XY) have shown that different lines have different tendencies to develop into particular types (Osafune et al., 2008). These characteristics could not be predicted on the basis of karyotypic sex alone (Cowan et al., 2004).

4. Hormonal Environment Interacts with Stem Cell Sex. The relationships between stem cells and hormones are complex—requiring consideration of the hormonal environment within which a cell or its ancestors developed as well as its current environment, whether in vitro or in vivo.

A review by Ray et al. (2008) demonstrates that sex hormones influence the characteristics of many types of stem cells, with effects that vary according to cell type:

When a patient’s own stem cells cannot be used therapeutically, success in stem cell transplantation depends on analyzing the interactions between: 1) the sex of donor cells used; 2) the sex of the host; 3) the type of stem cells transplanted; and 4) the illness being treated (see Method).

Discoveries about the interactions between species, stem cell sex, recipient sex, and hormonal and immunological variables in animal and in vitro research have prompted researchers to formulate questions relating to stem cell therapies for human patients. Currently, the only stem cell therapy in standard medical practice is hematopoietic stem cell (HSC) transplantation, used primarily to treat malignant disorders but also used in patients with immune deficiency or aplastic anemia (Gratwohl et al., 2010).

A study of 1,386 patients undergoing allogeneic HSC transplantation at a single medical center (about 75% for leukemias and the remainder for other conditions) showed that sex matching between donors and recipients correlated with better overall survival, although HSCs from male donors were associated with better long-term survival (Pond et al., 2006).

In pediatric leukemia, HSC transplantation from a female donor to a male recipient produces outcomes that are “unfavorable comparing with all other sex combinations” and “dismal in the presence of an MM (Human Leukocyte Antigen Mismatch).” Donor pregnancy was also found to interact with donor sex and recipient sex; when stem cells are derived from pregnant women donors and given to male patients, the risk of graft-versus-host disease increases (Gustaffson et al., 2004).

Donor and recipient sex also interact with the covariate of disease type—for example, when HSC transplantation is used to treat multiple myeloma, cells from female donors may produce better outcomes. Women patients who receive female HSCs have lower mortality than women patients treated with male HSCs. For men patients with multiple myeloma, the sex of donor cells did not significantly influence overall mortality, but did influence modes of mortality: Men patients treated with male HSCs were more likely to die from myeloma relapse, whereas men patients treated with female HSCs were more likely to die from non-relapse-related causes, such as graft-versus-host disease (Gahrton et al., 2005).

Systems for matching patients to donors for allogeneic HSC transplants now take donor sex and patient sex into account, along with numerous other variables, in order to optimize outcomes (Lee et al., 2007).

Conclusions

Researchers who reported and analyzed sex at the cellular level have identified sex differences in cell behavior that may be of relevance in developing therapeutics. These findings led researchers to investigate the causes of sex differences and discover both hormonal and genetic factors that govern stem cell behavior. In hematopoietic stem cell transplantation—the only stem cell therapy in widespread clinical use—clinicians have gathered data about interactions between donor sex, recipient sex, and other covariates in order to optimize donor-patient matching for allografts.

Next Steps

In basic research, scientists should be aware of the importance of sex as a variable and, in turn, identify the karyotype of cells used when reporting their research results. Results and null results should be reported (see Analyzing Sex). Reporting cell karyotype is important whether or not sex-based differences exist because this information permits secondary research reviews and meta-analyses. Granting agencies and journal editors can encourage such reporting through grant and publication guidelines.

A 2011 study at Mayo Clinic showed that the sex of the cell is not reported in most basic research (see chart). Not analyzing the sex of cells is money wasted. It is research lost to future meta-analysis.

Stem cell therapies hold great promise for treating debilitating diseases, such as Parkinson's disease and muscular dystrophy. Not taking the sex of the cells into account can lead to life-threatening consequences and leave researchers with unsolved puzzles. Take for example the problems an international collaboration between labs in Norway and Australia encountered working with bone marrow stem cells in mice. Researchers in the labs appropriately used both male and female mice (excellent research design), but they used all female stem cells without considering why. This is an unconscious decision that does not reflect best scientific practice. The result was that their male mice died, and they did not understand why.

Research has documented potential sex differences in the therapeutic capacity of stem cells. Muscle-derived stem cells, for example, show variability in proliferation and differentiation. Researchers found that XX cells showed a higher regenerative capacity than XY cells. This may constitute an important clinical finding, but requires further investigation.

Researchers should consider all combinations of donor/recipient sex interaction before ruling out sex as a variable. This type of donor/recipient analysis is also important in human organ transplant.

Gendered Innovation

Once the Norwegian and Australian labs considered all possible combinations of sex in donor/recipient interaction, they had greater success with their experiments. But research can't stop there. Other variables, such as stem cell type, the disease being treated, and hormonal and environmental factors, interact with sex to impact outcomes.